This invention relates to a new therapeutic composition, in particular an immunomodulating composition or a composition for uses in the treatment of immune deficiencies. The invention also discloses methods for the preparation of said composition and methods for the therapeutic application of the same.
Alpha-fetoprotein (AFP), a protein from mammalian fetal blood, has been held to be of a certain scientific interest since the moment it was first discovered in 1958. The biological properties of this protein are subject of numerous investigations: there is however still no final answer as to the role of this protein in the organism. It is known, that unsaturated fatty acids such as arachidonic and docosahexaenoic acid and their metabolites are characterized as natural AFP ligands and can be detected as complexes with the given protein circulating in blood vessels. However, AFP is not only a transport protein for unsaturated fatty acids. It has also been found that it forms complexes with bilirubin, retinoids and copper. In addition to the transport of low molecular weight substances AFP can take part in immune response regulation. Most of the studies on AFP""s immunoregulating properties indicate that the protein has immunosuppressing features.
It should also be pointed out, that practically no data on the immunosuppressing properties of AFP were obtained for the pure protein, but using AFP-enriched sera or amniotic fluid during in vitro experiments. The immunoregulating properties of AFP were shown to be dependent on the origins of the preparations and the purification methods. For example, AFP obtained from fetal liver is characterized by a stronger suppression of mitogene-induced lymphocyte transformation in comparison with AFP, obtained from human blood of patients suffering from primary liver cancer.
At the same time it has been demonstrated, that AFP stimulates tissue regeneration after injuries. AFP tended to decrease the inflammatory processes artificially simulated in animals supposedly by blocking the receptors of immunocompetent cells. It has also recently been shown in a series of experiments, that AFP is actively absorbed by growing and differentiating cells, and this process is controlled by the quantity of expressed AFP-receptors. It was found that intracellular AFP concentration increased simultaneously with the increase of the quantity of AFP-receptors on the surface of proliferating T-lymphocytes and malignant cells. It was stated, based on these data, that this protein functions as a shuttle-transporter, bringing the ligand inside the cell and then returning into intercellular liquid to repeat the cycle (Esteban C., et al., Int. J. Cancer, v.49, p. 425-430, 1991).
The most important ligands transported inside the cell are unsaturated fatty acids, namely arachidonic and docosahexaenoic acid and their metabolites. It is experimentally proven, that the presence of AFP significantly increases the flow of these acids into the cytoplasm of activated T-lymphocytes (Torres J. M., et al., J. Cell. Physiol, v. 150, p.456-462, 1992).
The increase of the concentration of unsaturated fatty acids is of great importance as said acids are not only necessary structural components of the cell membrane, but also serve as an additional source of energy for the cells. The metabolites of these acids, in particular those of arachidonic acid, can act as secondary messengers, thus participating in the regulation of cellular growth and differentiation (Bevan S., et al., Nature (London), v. 328, p. 20, 1987).
Anandamide (arachidonyl-2-ethanolamid) is one of the recently discovered fatty acid metabolites. It is characterized by the high physiological effect targeted to brain. Anandamide is a novel lipid neurotransmitter first isolated from porcine brain. It has been shown to be a functional agonist for cannabinoid CB1 and CB2 receptors. Its presence results in many pharmacological effects caused by delta 9-tetra-hydrocannabinol (delta 9-THC). Anandamide parallels delta 9-THC in its specific interaction with the cannabinoid receptor and in the inhibition of adenylate cyclase. For many decades the mechanism of action of cannabinoid compounds, which are structurally similar to delta 9-THC, was unknown. Tremendous progress has recently been made in characterising cannabinoid receptors both centrally and peripherally as well as in studying the role of the second messenger systems at cellular level. Cannabinoid derived drugs have been used for centuries for medicinal purposes. However, these drugs on the market today lack specificity and produce many side effects (Chakrabarti A., et al., Brain. Res. Bull., v.45, 30 p.67-74, 1998).
Anandamide can be formed enzymatically via two separate synthetic pathways in the brain: enzymatic condensation of the free arachidonic acid and ethanolamine; and formation of N-arachidonoyl phosphatidylethanolamine from phosphatidyletanolamine and arachidonic acid esterified at the 1-position of phosphatidylcholine, and subsequent release of anandamide from N-arachidonoyl phosphatydylethanolamine through the action of a phosphodiesterase (phospholipase D) (Suguira T., et al., Eur. J. Biochem., v.240. p.53-62, 1996).
N-acyl-transferase catalyses the transfer of arachidonoyl residue onto the NH2 group of phosphatydylethanolamine. This enzyme is Ca2+dependent and is mostly localised in brain and testis. The pathway of anandamide formation is presented below: 
Rxe2x80x94Arachidonoyt
R1 and R2xe2x80x94alkyl
N-arachidonoyl phosphatidylethanolamine could also be a substrate for phospholipase C (Brockerhoff H., Jensen R. G., Lipolytic enzymes, Academic press, New York-San Francisco-London, 1974). In this case the enzymatic reaction results in formation of N-arachidonoyl aminoethylphosphate (N-AAP). 
The absence of literature data on N-AAP presence in brain supports the assumption, that this phosphate is unstable and can be quickly transformed to anandamide by endogenous phosphatases during processing of the brain preparations. To study the N-AAP biological activity one has to consider a reversible complex of AFP with N-AAP. In this case N-AAP can be protected by the protein molecule from the enzymatic influence in blood vessels as well as in the other biological liquids.
The idea to use reversible complexes of transport proteins with the conjugates of their natural ligands with drugs to strengthen their pharmaceutical effect and reduce the side effects, particularly during cancer treatment, was first reported in 1958 (Mathe G. et al., C. R. Seances Acad. Sci. v. 246, p. 1626-1628, 1958; Magnenat R. et al., Eur. J. Cancer., v. 5. p. 3340, 1969). The reversible complex of AFP with the conjugates of daunomicin with arachidonic and docosohexaenoic acids appeared to be more effective cytostatic agent for hepatoma AH-66 cells, generating more AFP than the free daunomicin (Deutsch H. F. et al., Cancer Res. v. 43, p.2668-2662, 1983), and the conjugate of 2-deoxy-5-fluorouridine-oleic and docoso hexaenoic acids with AFP had much greater cytotoxic activity for cancer cell lines H 1-29, than the free 2-deoxy-5-fluorouridine (Halmos T. et al., Biochem. Pharmacol v. 44., p. 149-156, 1992).
Some direct immunologic response observations on the role of a factor that appears to be AFP have been reported (Abramsky, O., et al., Isr. Med., vol. 15, p. 943, 1979; Brenner T., et al., Immunol. Lett., vol. 3, p. 163, 1981). They found that what is likely to be fetal AFP prevented the development of myasthenia gravis in rabbits and, furthermore, that clinical signs of the disease in these animals disappeared when they were treated with the assumed AFP. It was shown that experimental allergic encephalomyelitis induced in guinea pigs was successfully treated as well as partially prevented by administration of AFP (Abramsky O., et al., J. Neuroimmunol., vol. 2, p. 1, 1982).
It has thus been shown that AFP, depending on its origin and surrounding conditions, exerts different functions by different mechanisms. Firstly, there is a regulatory effect on the concentration of the unbound form of its various ligands (e.g. fatty acids, estrogens, phytosteroids). It is known, that fatty acids, in particular polyunsaturated fatty acids, modulate positively or negatively many steps of the action of various steroids and many enzymes involved in the transduction of membrane-triggered signals. Secondly, different conformations (holoforms) of AFP, depending on the nature and concentration of the ligand(s) bound to it, might influence the binding of the protein to specific receptor(s) and as a consequence influence it""s/their biological properties (internalisation, action on the membrane signal transduction pathway). Thirdly, in addition to the mechanisms proposed above, the protein can exert effects associated with other signals, such as growth factors.
Obviously, there appears to be no uniform and consistent understanding of the mechanisms of AFP. Presently used immunomodulating substances and in particular immunostimulating substances are not without their drawbacks. Interferon preparations give influenza-like symptoms in about 90% of the patients and the risk of other side effects must be considered. Typically, the side effects range from muscle and skeletal soreness and pains, headache and similar symptoms to more serious symptoms as leucopenia, anaemia, trombocytopenia, splenomegalia and hepatomegalia, just to mention some examples.
The aim of the present invention is to make available a new pharmaceutical immunomodulating composition exhibiting improved properties, not only with respect to therapeutical properties such as efficacy and extent of application, but also pharmacological and technical properties such as ease of manufacture, storage, mixing and administration.
The present invention concerns a therapeutically useful complex, in particular an immunomodulating complex according to the attached claims. The invention will be described in closer detail in the following description and examples.
The invention makes available an equilibrium reversible complex of alpha-fetoprotein and N-arachidonoyl aminoethylphosphate, in particular an equilibrated non-covalent complex of N-arachidonoyl aminoethylphosphate (N-AAP), a metabolite of arachidionic acid and alpha-fetoprotein (AFP) of high purity, for example AFP isolated from human cord blood with more than 99% purity. The chemical structure of the non-protein part of the complex is presented below:
CH3(CH2)4(CHxe2x95x90CHCH2)4CH2CH2CONHCH2CH2OPO(OH)2 
The inventive complex may contain its components in highly varying molar ratios, such as from an equimolar ratio to a significant overabundance of N-AAP in relation to AFP. Normally, the complex contains from 1 up to 300 moles N-AAP per mole AFP. The inventive complex may be obtained by adding an ethanol solution of N-AAP to a diluted water solution of AFP followed by ultrafiltration, said filtration resulting in concentrating the solution and removing the N-AAP that remained unbound to AFP. The AFP concentration in solution varies from 0.1 up to 2 mg/ml and that of N-AAPxe2x80x94from 0.005 up to 30 mg/ml.
In the inventive complex, the protein is reversible linked not to one or several molecules of the ligand but surprisingly with a micelle, containing up to 300 molecules of N-AAP. It is known, that the natural AFP ligands like arachidonic, docosohexaenoic acids, etc. are sparingly soluble in water. If a concentrated ethanol solution of these substances is injected into water under special conditions one obtains a colloid solution. The obtained colloid particles (micelles) contain about from 50 to about 300 or more molecules of the lipid. The addition of unsaturated fatty acidsxe2x80x94AFP ligands to AFP water solution results in the formation of protein-lipid complexes. The properties of such complexes have been insufficiently studied, but it is however possible to assume, that their formation occurs not only due to the hydrophobic fragment of the protein molecule but is also stipulated by the participation of AFP""s active centre(s). Attempts to produce AFP complexes with other fatty acids, not being the ligands to this protein, namely with other fatty acids, have been unsuccessful. The changes of molecular weight of the protein as judged by gel-filtration is an evidence of the existence of AFP complexes with the micelles of its natural ligands.
In one embodiment, the molecular weight of AFP incorporated in the complex with its natural ligand or its derivative (for example N-AAP) increases by approx. 2 times, while gel-filtration of AFP with palmitoyl acid micelles did not result in the changes of elution volume in comparison with that for free AFP. The micelle contained about 200-300 molecules of lipid. The obtained preparations of AFP complexes with the micelles of its natural ligands are characterized as reversible protein-lipid complexes, but at the same time have the properties of proteoliposomes (Degrip W. J. Biochem J. Mar. 1. 330, p. 667-674, 1998).
In another embodiment, the molecular weight of AFP incorporated in the complex with its natural ligand or its derivative (for example N-AAP) increases by approx. 2-3 times. The micelles contained 100-300 molecules of lipid. The obtained preparations of AFP complexes with the micelles of its natural ligands or metabolites are characterised as reversible protein-lipid complexes, but at the same time have the properties of proteoliposomes.
It has been shown that AFP enter the cells via small vesicles and endosomes and move to multivesicular bodies and tubular vesicular elements located in the Golgi-centrosphere region to be finally recycled back into the medium (Geuskens M., et al. Microsc. Res. Tech. v.28, p. 297-307, 1994).
Based on the literature data and the experimental results obtained by the present inventor it is suggested, that the reversible complexes of AFP with N-AAP penetrates into lymphocytes by means of AFP""s receptor intermediated endocytosis. On the one hand, the AFP/N-AAP complex inside lymphocytes could apparently regulate the synthesis of phospholipids as the structural components of cellular membrane. On the other hand N-AAP is a source of arachidonic acid which is further being incorporated into the phospholipid structures.
The influence of AFPIN-AAP complexes as well as their basic components on humoral immune response was estimated by counting the quantity of antibody-forming cells (AFC) in the spleen. It has been experimentally proved that N-AAP in itself does not exhibit immunogenic activity. The relative amount of AFC cells on the 5-th day after N-AAP injection to the animals immunized with sheep erythrocytes was not significantly changed in comparison with control series. Administration of the same dosage of the inventive AFP/N-AAP complex (AFPIN-AAP ratio 1:200) resulted in that the relative amount of AFC increased 87% and the total AFC amount increased 162% on the 5-th day after injection in comparison with the amount of cells in animals immunized only with sheep erythrocytes.
An administration of the inventive complex in AFP/N-AAP ratios of 1:100 or 1:300 showed a slightly decreased immunostimulating activity of the complex. On the 5-th day after injection of a 1:100 complex the apparent AFC amount had increased by 30%, and the total amount by 79%. For a 1:300 complex the increase in apparent AFC amount was 48.3%, and for the total cell amount 103.3%. The results show a significant effect of the complex within the interval 1:100-1:300, with an improved effect corresponding to the AFP/N-AAP ratio 1:200. However, the AFP/N-AAP ratio can be varied within a broader interval, e.g. 1:1-1:10000.
AFP alone, administered in corresponding doses reduces or does not significantly effect the immunogenic characteristics of sheep erythrocytes in mice.
AFP was isolated from human cord blood by immunoaffinity chromatography on monoclonal antibodies against AFP immobilised on Sepharose(copyright), immunoaffinity chromatography on polyclonal antibodies to the proteins of normal human blood and gel-filtration on Sephacryl(copyright) S-200. The AFP preparation thus obtained was more than 99% purity and did not contain low molecular weight impurities and retained completely its biological activity.
Other sources of AFP may be purified and/or modified AFP from other mammals, for example from genetically modified mammals, or from cell cultures. Preferably, the AFP is biotechnologically manufactured using a cell culture of genetically modified cells expressing human AFP. With knowledge of the nucleotide sequence coding for human AFP, this can be inserted in a host, together with necessary promoters and other sequence information, for example sequences influencing the extracellular expression of AFP. The AFP is collected from the cell culture and purified by chromatography, and may be further purified by gelfiltration. In any case, the production method must involve steps, which guarantee that the final product is free from pyrogens and possible viral or bacterial contaminants. Suitable production methods can for example be found in the field of interferon production.
According to an embodiment of the invention, the AFP/N-AAP complex is used as a therapeutic agent either as such, or used for the manufacture of a therapeutical preparation, possibly containing other agents. The inventive complex is particularly suitable as an immunostimulating agent, e.g. for the treatment of immune deficiencies. The complex can also be used for the manufacture of an immunostimulating preparation.
According to a preferred embodiment, the inventive complex is used for the treatment of immune disorders associated with cancer therapy. The inventive complex can also be used for the manufacture of a pharmaceutical preparation for the treatment of cancer. Examples of such disorders or immune deficiencies occurring as a consequence of cancer treatment, include neutropenia.
The inventive complex can also be used as a prophylactic agent in patients susceptible for infections, or for the manufacture of a pharmaceutical composition for the treatment of such patients.
Consequently, the invention also concerns methods for the treatment of immune deficiencies, wherein an equilibrium reversible complex according to the present invention is administered to a mammal. Preferably, said complex is administered intravenously.
Medical form preparation: The active composition may be administered intravenously. Alternatively, to simplify storage and handling, the composition can be prepared as a sterile powder for extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent allowing for easy handling by syringe and similar devices. Further, the preparation must be stable under conditions of manufacture and storage and must be protected against the contaminating action of micro-organisms such as bacteria and fungi.
Sterile injectable solutions are prepared by incorporating AFP and N-AAP in a required amount of water, ultrafiltration (concentration) of the solution followed by filter sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying or suitable freeze-drying techniques that yield a powder of the active ingredients from previously sterile filtered solutions thereof.
For direct introduction of a complex to patients a sterilized preparation is first injected in a physiological saline solution (100-500 ml) or in a protein (albumin) solution then intravenously administered to a patient. When a lyophilized preparation is used, it should be previously dissolved in 2-10 ml of sterilized solution (distilled water, physiological saline solution or albumin solution) whereupon the obtained sterilized solution is added to 100-500 ml physiological solution or protein solution for intravenous introduction, same as in case of using the solutions.
The therapeutic effective doses are in interval from 2 mg/kg to 7 mg/kg for N-AAP and from 0.2 mg/kg to 0.7 mg/kg for AFP.